Cloning, Overexpression, and Functional Characterization of a Phytase from the Genus Bacillus

Abstract

Bacillus strain WYCQ02 was obtained from soil samples by high-temperature screening. A 1,327-bp DNA fragment containing a 1,152-bp long open reading frame named phyC-WYCQ02 was amplified from the genomic DNA of strain WYCQ02 by PCR. The ORF encoded a polypeptide of 383 amino acid residues with a putative signal peptide of 26 amino acids. The 1,089-bp fragment encoding the mature peptide of neutral phytase and a 6 × histidine tag was cloned into the plasmid pPIC9K. The expression vector, pPIC9K-phyC, was linearized and transformed in Pichia pastoris. The molecular weight of phytase was estimated to be approximately 53 kDa by electrophoresis. The optimal temperature of the purified phytase was 55°C and the optimal pH value was between 7.0 and 8.0. After incubation at 70°C for 10 and 30 min, the relative activity was still over 80 and 62%, and over 70% of enzyme activity remained in the pH range of 5.0-10.0. There was no significant difference in enzymatic activity after incubation for 30 or 60 min in buffer with different pH values, therefore the purified phytase had some acid and alkali resistance. The phytase gene and the engineered yeast strain may have value in industrial applications.